Conjugation of Zika virus EDIII with CRM197, 8-arm PEG and mannan for development of an effective Zika virus vaccine.

Zika virus (ZIKV) induces neurological and autoimmune complications such as microcephaly and Guillain-Barre syndrome. Effective vaccines are necessary to prevent the ZIKV infection. E protein of ZIKV is responsible for virus attachment, entry, and fusion. The domain III of E protein (EDIII) contains the neutralizing epitopes and is ideal to act as an antigen for ZIKV vaccine. However, EDIII is poorly immunogenic. CRM197 is a carrier protein and can activate T helper cells for EDIII. Mannan is a ligand of TLR-4 or TLR-2. Eight-arm PEG can link multiple EDIII molecules in one entity. In the present study, EDIII was covalently conjugated with CRM197, 8-arm PEG and mannan to improve the immunogenicity of EDIII. The conjugate (CRM-EDIII-PM) elicited high EDIII-specific antibody titers in the BALB/c mice. Th1-type cytokines (IFN-γ and IL-2) and Th2-type cytokines (IL-5 and IL-10) were secreted at a marked level. Thus, CRM-EDIII-PM could stimulate potent humoral and cellular immune response to EDIII. The serum exposure of CRM-EDIII-PM to the immune system was prolonged. Moreover, CRM-EDIII-PM did not lead to apparent toxicity to the organs. Therefore, CRM-EDIII-PM was expected as a promising vaccine candidate for its ability to induce strong immune responses.

Engineered Nanoparticle Applications for Recombinant Influenza Vaccines.

Influenza viruses cause seasonal epidemics and represent a pandemic risk. With current vaccine methods struggling to protect populations against emerging strains, there is a demand for a next-generation flu vaccine capable of providing broad protection. Recombinant biotechnology, combined with nanomedicine techniques, could address this demand by increasing immunogenicity and directing immune responses toward conserved antigenic targets on the virus. Various nanoparticle candidates have been tested for use in vaccines, including virus-like particles, protein and carbohydrate nanoconstructs, antigen-carrying lipid particles, and synthetic and inorganic particles modified for antigen presentation. These methods have yielded some promising results, including protection in animal models against antigenically distinct influenza strains, production of antibodies with broad reactivity, and activation of potent T cell responses. Based on the evidence of current research, it is feasible that the next generation of influenza vaccines will combine recombinant antigens with nanoparticle carriers.

Comprehensive evaluation of chitosan nanoparticle based phage lysin delivery system; a novel approach to counter S. pneumoniae infections.

Cpl-1, an endolysin derived from Cp-1 phage has been found to be effective in a number of in-vitro and in-vivo pneumococcal infection models. However its lower bioavailability under in-vivo conditions limits its applicability as therapeutic agent. In this study, Cpl-1 loaded chitosan nanoparticles were set up in order to develop a novel therapeutic delivery system to counter antibiotic resistant S. pneumoniae infections. Interactions of chitosan and Cpl-1 were studied by in-silico docking analysis. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were prepared by using ionic gelation method and the process was optimized by varying chitosan:TPP ratio, pH, stirring time, stirring rate and Cpl-1 concentration. Chitosan nanoparticles and Cpl-1 loaded chitosan nanoparticles were characterized to ascertain successful formation of nanoparticles and entrapment of Cpl-1 into nanoparticles. Chitosan nanoparticles and Cpl-1 loaded nanoparticles were also evaluated for nanoparticle yield, entrapment efficiency, in-vitro release, stability, structural integrity of Cpl-1, in-vitro bioassay, swelling studies, in-vitro biodegradation and heamolysis studies. Mucoadhesion behavior of chitosan nanoparticles and Cpl-1 loaded nanoparticles was explored using mucous glycoprotein assay and ex-vivo mucoadhesion assay, both preparations exhibited their mucoadhesive nature. Cellular cytotoxicity and immune stimulation studies revealed biocompatible nature of nanoparticles. The results of this study confirm that chitosan nanoparticles are a promising biocompatible candidate for Cpl-1 delivery with a significant potential to increase bioavailability of enzyme that in turn can increase its in-vivo half life to treat S. pneumoniae infections.

RVG29-modified docetaxel-loaded nanoparticles for brain-targeted glioma therapy.

Gliomas are the most common malignant brain tumor, but treatment is limited by the blood-brain barrier (BBB), especially for chemotherapeutic drugs. Although some chemotherapy drugs can pass through the BBB, many of these agents are toxic to normal brain tissue. To maximize therapeutic effects, chemotherapeutic drugs must accumulate at the glioma site. In this study, a specific ligand (the RVG29 peptide) that can combine with acetylcholine receptors was conjugated to polyethylene glycol-modified poly-(d,l-lactide-co-glycolide) (PEG-PLGA) to develop a targeted carrier; preparation of the targeted docetaxel nanoparticles (DTX-NPs) was performed by the nanoprecipitation method. The NPs were approximately 110 nm and had smooth surfaces. Enzyme-linked immunoassay results showed that the amount of receptor on the surface of glioma cells was 2.04-fold higher than that of nonmalignant cells, which may promote accumulation of RVG29-modified NPs at the targeting site. NPs showed targeting properties for glioma cells compared with the non-targeting NPs in an in vitro cellular uptake test. Targeted NPs also showed better BBB penetration in an in vitro model. In vivo tests indicated that RVG29-PEG-PLGA-NPs could selectively accumulate in intracranial glioma tissue. In conclusion, these results indicated that the RVG29-modified NPs have potential efficacy for glioma therapy.

Targeted transport of nanocarriers into brain for theranosis with rabies virus glycoprotein-derived peptide.

For successful theranosis of brain diseases, limited access of therapeutic molecules across blood-brain barrier (BBB) needs be overcome in brain delivery. Currently, peptide derivatives of rabies virus glycoprotein (RVG) have been exploited as delivery ligands to transport nanocarriers across BBB and specifically into the brain. The targeting peptides usually conjugate to the nanocarrier surface, and the cargoes, including siRNA, miRNA, DNA, proteins and small molecular chemicals, are complexed or encapsulated in the nanocarriers. The peptide ligand of the RVG-modified nanocarriers introduces the conjugated targeted-delivery into the brain, and the cargoes are involved in disease theranosis. The peptide-modified nanocarriers have been applied to diagnose and treat various brain diseases, such as glioma, Alzheimer's disease, ischemic injury, protein misfolding diseases etc. Since the targeting delivery system has displayed good biocompatibility and desirable therapeutic effect, it will raise a potential application in treating brain diseases.

Cytosolic delivery of siRNA by ultra-high affinity dsRNA binding proteins.

Protein-based methods of siRNA delivery are capable of uniquely specific targeting, but are limited by technical challenges such as low potency or poor biophysical properties. Here, we engineered a series of ultra-high affinity siRNA binders based on the viral protein p19 and developed them into siRNA carriers targeted to the epidermal growth factor receptor (EGFR). Combined in trans with a previously described endosome-disrupting agent composed of the pore-forming protein Perfringolysin O (PFO), potent silencing was achieved in vitro with no detectable cytotoxicity. Despite concerns that excessively strong siRNA binding could prevent the discharge of siRNA from its carrier, higher affinity continually led to stronger silencing. We found that this improvement was due to both increased uptake of siRNA into the cell and improved pharmacodynamics inside the cell. Mathematical modeling predicted the existence of an affinity optimum that maximizes silencing, after which siRNA sequestration decreases potency. Our study characterizing the affinity dependence of silencing suggests that siRNA-carrier affinity can significantly affect the intracellular fate of siRNA and may serve as a handle for improving the efficiency of delivery. The two-agent delivery system presented here possesses notable biophysical properties and potency, and provide a platform for the cytosolic delivery of nucleic acids.

Preferential targeting of disseminated liver tumors using a recombinant adeno-associated viral vector.

A novel selectively targeting gene delivery approach has been developed for advanced hepatocellular carcinoma (HCC), a leading cause of cancer mortality whose prognosis remains poor. We combine the strong liver tropism of serotype-8 capsid-pseudotyped adeno-associated viral vectors (AAV8) with a liver-specific promoter (HLP) and microRNA-122a (miR-122a)-mediated posttranscriptional regulation. Systemic administration of our AAV8 construct resulted in preferential transduction of the liver and encouragingly of HCC at heterotopic sites, a finding that could be exploited to target disseminated disease. Tumor selectivity was enhanced by inclusion of miR-122a-binding sequences (ssAAV8-HLP-TK-122aT4) in the expression cassette, resulting in abrogation of transgene expression in normal murine liver but not in HCC. Systemic administration of our tumor-selective vector encoding herpes simplex virus-thymidine kinase (TK) suicide gene resulted in a sevenfold reduction in HCC growth in a syngeneic murine model without toxicity. In summary, we have developed a systemically deliverable gene transfer approach that enables high-level expression of therapeutic genes in HCC but not normal tissues, thus improving the prospects of safe and effective treatment for advanced HCC.

Enhanced BBB permeability of osmotically active poly(mannitol-co-PEI) modified with rabies virus glycoprotein via selective stimulation of caveolar endocytosis for RNAi therapeutics in Alzheimer's disease.

RNA interference (RNAi) holds one of the promising tools for Alzheimer's disease (AD) treatment by directly arresting the causative genes. For successful RNAi therapeutics for AD, limited access of therapeutic genes to the brain needs to be overcome by developing siRNA delivery system that could cross the blood-brain barrier (BBB). Here, we report a non-viral vector, rabies virus glycoprotein (RVG)-modified poly(mannitol-co-PEI) gene transporter (PMT), R-PEG-PMT. The RVG ligand directed the PMT/siRNA complexes toward the brain through binding to nicotinic acetylcholine receptors expressed on BBB. In mechanistic study using in vitro BBB model, we observed that osmotically-active PMT enhanced the receptor-mediated transcytosis by stimulating the caveolar endocytosis. The potential of RNAi therapeutics for AD using R-PEG-PMT/siBACE1 complexes was demonstrated in vitro and in vivo. Our results suggest that R-PEG-PMT is a powerful gene carrier system for brain targeted RNAi therapeutics with synergistic effect of RVG ligand and PMT on well-modulated receptor-mediated transcytosis through BBB.

In vivo bacteriophage peptide display to tailor pharmacokinetics of biological nanoparticles.

PURPOSE: Clinical use of most radiolabeled targeting agents has been limited because of the uptake and retention in kidney and/or liver. We hypothesized that bacteriophage (phage) display could be exploited to select for peptide sequences with fast clearance and low kidney uptake with the added ability to redirect phage clearance away from the reticuloendothelial system towards the kidney possessing rapid kidney clearance. PROCEDURES: In vivo phage display was performed to identify peptides displayed on phage that were excreted rapidly into the urine of mice. A novel in vitro assay using kidney cells, developed to predict in vivo kidney retention, and in vivo pharmacokinetic analyses were performed to characterize selected peptides/phage clones. RESULTS: Forty-three renal clearance clones (RCC) were identified. In vivo mixing experiments and in vitro kidney cell assays identified RCC1-02 as the lead compound. In vivo analysis of fluorescently labeled phage clones demonstrated the ability of RCC1-02 peptide to redirect the biodistribution of the large phage particle towards excretion via the kidney. Pharmacokinetic analysis of [(111)In]-radiolabeled peptides revealed that kidney retention of the control ErBB-2-avid peptide, [(111)In]DOTA-KCCYSL, at 2-h postinjection was 5.7 ± 0.7 %ID/g. In comparison, [(111)In]DOTA-RCC1-02 had kidney retention values of 1.66 ± 0.43 %ID/g, respectively. CONCLUSIONS: In vivo phage display can identify phage and corresponding peptides that rapidly clear the renal system. In the future, these peptides may be used to impart favorable pharmacokinetics onto a wide range of radioimaging or therapeutic macromolecules.

Distribution characteristics of DNA vaccine encoded with glycoprotein C from Anatid herpesvirus 1 with chitosan and liposome as deliver carrier in ducks.

BACKGROUND: A eukaryotic expression plasmid encoding glycoprotein C (gC) of Anatid herpesvirus 1 (AnHV-1) (pcDNA3.1-gC) was constructed and validated. The tissue distribution of chitosan/DNA complexes, liposome/DNA complexes and pcDNA3.1-gC alone were evaluated using a quantitative real-time PCR based TaqMan™ probe following intramuscular administration in ducklings. RESULTS: Compared with pcDNA3.1-gC alone, liposomes universally increased the plasmid DNA copy number at the injection sites, liver, spleen, heart, brain, bursa of Fabricius, and especially in the enteron (esophagus, duodenum, rectum, and cecum). Chitosan also universally increased the plasmid DNA copy number at the injection sites, liver, spleen, heart, brain and esophagus. Compared with lipoplex-gC, higher chitosan-gC plasmid DNA copy numbers were detected at the injection sites, liver, spleen, heart, brain and esophagus. In contrast, compared with lipoplex-gC, lower copy numbers of chitosan-gC plasmid DNA were detected in the duodenum, rectum and cecum. CONCLUSIONS: The results of this study demonstrated that chitosan and liposomes mediated rapid and extensive plasmid distribution in duck tissues, with low levels maintained from 1 d after DNA vaccination.

Nanoglue: an alternative way to display cell-internalizing peptide at the spikes of hepatitis B virus core nanoparticles for cell-targeting delivery.

Cell-internalizing peptides (CIPs) can be used to mediate specific delivery of nanoparticles across cellular membrane. The objective of this study was to develop a display technique using hepatitis B virus (HBV) capsid-binding peptide as a "nanoglue" to present CIPs on HBV nanoparticles for cell-targeting delivery. A CIP was selected from a phage display library and cross-linked specifically at the tips of the spikes of the HBV capsid nanoparticle via the "nanoglue" by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS). Fluorescent oligonucleotides packaged in the nanoparticles and the fluorescein molecules conjugated on the nanoparticles were delivered to cells by using this display technique. This study demonstrated a proof of principle for cell-targeting delivery via "nanoglue" bioconjugation.

Central nervous system distribution of the poxviral proteins after intranasal administration of proteins and titering of vaccinia virus in the brain after intracranial administration.

Poxviral proteins are known to interact with the immune system of the host. Some of them interact with the transcription factors of the host, whereas others interact with the components of the immune system. Vaccinia virus secretes a 28.8-kDa complement control protein (VCP), which is known to regulate the complement system. This protein helps the virus to evade the immune response of the host. Such viral proteins might also prove beneficial in the treatment and prevention of the progression of the disorders, where up-regulation of the complement system is evident. VCP has been shown experimentally to be effective in protecting tissues from inflammatory damage in the rodent models of Alzheimer's diseases (AD), spinal cord injury, traumatic brain injury, and rheumatoid arthritis. Not only VCP, but also other poxviral proteins could be used therapeutically to treat or prevent the progression of the brain disorders, where the immune system is inadequately controlled. However, being a protein that cannot traverse the brain barrier because of its size, delivery of such proteins to the central nervous system (CNS) could be a limiting factor in their usefulness as CNS therapeutics. In this chapter, we show methods for the intranasal route of administration of a protein and show ways to detect its distribution in the cerebrospinal fluid (CSF) and to the different parts of the brain. These protocols can be extended to examine the distribution of viral antigens in the brain. A protocol is also included to quantitate vaccinia virus in different segments of the brain after intracranial administration of the virus.

Intracellular delivery: exploiting viral membranotropic peptides.

Recent advances in the understanding of cellular and molecular mechanisms of the pathogenesis of several diseases offer the possibility to address novel molecular targets for an improved diagnosis and therapy. In fact, in order to fulfill their function, macromolecular drugs, reporter molecules, and imaging agents often require to be delivered into specific intracellular compartments, usually the cytoplasm or the nucleus. From a medical perspective, biological membranes represent a critical hindrance due to their barrier-like behaviour not easily circumvented by many pharmacologically-active molecules. Therefore, identifying strategies for membrane translocation is essential. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs). These peptides, which are able to efficiently translocate macromolecules through the plasma membrane, have attracted a lot of attention, and new translocating peptides are continuously described. In this review, we will focus on the viral derived peptides, and in particular those derived by viral entry proteins that may be useful as delivery vehicles due to their intrinsic properties of inducing membrane perturbation.

Targeted delivery of proteins into the central nervous system mediated by rabies virus glycoprotein-derived peptide.

PURPOSE: Delivery of therapeutic proteins across the blood-brain barrier (BBB) is severely limited by their size and biochemical properties. Here we showed that a 39-amino acid peptide derived from the rabies virus glycoprotein (RDP) was exploited as an efficient protein carrier for brain-targeting delivery. METHODS: Three proteins with different molecular weight and pI, ß-galactosidase (ß-Gal), luciferase (Luc) and brain-derived neurotrophic factor (BDNF), were fused to RDP and intravenously injected into the mice respectively. The slices of different tissues with X-Gal staining were used to examine whether RDP could deliver ß-Gal targeted into the CNS. The time-course relationship of RDP-Luc was studied to confirm the transport efficiency of RDP. The neuroprotective function of RDP-BDNF was examined in mouse experimental stroke to explore the pharmacological effect of RDP fusion protein. RESULTS: The results showed that the fusion proteins rapidly and specific entered the nerve cells in 15 min, and the t(1/2) was about 1 hr. Furthermore, RDP-BDNF fusion protein showed the neuroprotective properties in mouse experimental stroke including reduction of stroke volume and neural deficit. CONCLUSIONS: RDP provides an effective approach for the targeted delivery of biological active proteins into the central nervous system.

[Zebrafish as a model animal for the study of blood-brain barrier permeability by biomolecules].

Blood-brain barrier (BBB) is the major obstacle for drug delivery into the central nervous system (CNS). However, there is no ideal model animal for the study of BBB permeability till now. Currently zebrafish (Danio rerio) has emerged as a powerful model organism for the study of vertebrate biology. In this study, the feasibility of using zebrafish as model animal was investigated for BBB permeability by comparing the results of administration of BBB-penetrating peptide and protein to mouse and zebrafish. The results showed that the BBBs of mouse and zebrafish were similar in molecular permeability. Additionally, zebrafish has advantageous features as a model animal, such as small size, fertile and easy to breed. Therefore, it is suggested that zebrafish may be a favored model for the study of BBB permeability.

[Trafficking pathway and affectivity on extracellular signal-regulated kinase phosphorylation of anti-tumor fusion protein of epidermal growth factor-adenovirus early region 4 open reading frame 4 protein].

OBJECTIVE: To investigate the intracellular trafficking pathway of fusion protein epidermal growth factor-adenovirus early region 4 open reading frame 4 protein (EGF-E4orf4) internalized via epidermal growth factor (EGF) receptor and its affectivity on extracellular signal-regulated kinase (ERK) phosphorylation. METHODS: MDA-MB-231 and BGC823 cells were incubated with fluorescein isothiocyanate-EGF-E4orf4 or EGF at different time points. The specific molecular mark of early endosome or late lysosome was labeled by indirect immunofluorescence, and then colocalization staining was observed using confocal laser microscopy. The levels of ERK phosphorylation were detected by Western blot. RESULTS: The fluorescent signal of fusion protein EGF-E4orf4 accumulated within the cells and congregated to the perinuclear region. A nucleus localization of the fusion protein was only at MDA-MB-231 cell. Colocalization of EGF-E4orf4 with early endosome/late lysosome was observed. EGF-E4orf4 stimulated ERK phosphorylation, which was most obvious 10 minutes after stimulation, and then gradually attenuated, which was similar to EGF stimulation but with a less decrease. CONCLUSIONS: Internalized EGF-E4orf4 can be slowly degraded via endosome-lysosome pathway. The action features of EGF-E4orf4 are remarkably different between MDA-MB-231 and BGC823 cells, which may help explain the differences in its anti-tumor potency and in the special selectivity toward different tumor cells.

Controlled recovery of the transcription of nanoparticle-bound DNA by intracellular concentrations of glutathione.

Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) bind DNA through complementary electrostatic interactions, resulting in complete inhibition of DNA transcription of T7 RNA polymerase. DNA was released from the nanoparticle by intracellular concentrations of glutathione, resulting in efficient transcription. The restoration of RNA production was dose-dependent in terms of GSH, with considerable control of the release process possible through variation in monolayer structure. This work presents a new approach to controlled release of DNA, with potential applications in the creation of transfection vectors and gene regulation systems.

Vaccinia virus complement control protein diminishes formation of atherosclerotic lesions: complement is centrally involved in atherosclerotic disease.

Complement is known to be activated in atherosclerotic lesions, but the importance of this event in disease pathology is a matter of debate. Studies of rabbits fed a high-fat diet have indicated complement activation as a rate-limiting step, whereas results from genetically modified mouse strains (ApoE-/- or LDLR-/-) have failed to support this finding. To resolve whether this reflects differences between species or between genetically driven and diet-induced disease, we studied the effect of a complement inhibitor, vaccinia virus complement control protein (VCP), on C57BL/6 mice, the background strain of ApoE-/- and LDLR-/- mice. Atherosclerosis was induced by a high-fat diet, and VCP (20 mg/kg) was injected once per week after the eighth week. Fatty streak development was monitored at 15 weeks by microscopic examination of oil red-O-stained sections from the root of the aorta. VCP injections led to significant (50%) reduction of lesion size (P = 0.004). Lesions were marked by gradual accumulation of lipids and macrophages but did not develop beyond the fatty streak stage. VCP activity disappeared from serum in 4 days, and the possibility therefore exists that a higher level of protection may be achieved by more frequent injections. We conclude that the development of fatty streaks in diet-induced atherosclerotic disease can be significantly retarded by prophylactic treatment with a complement inhibitor. These results support previous findings from complement-deficient rabbits and suggest that the pathogenesis of atherosclerosis in diet-induced disease differs from that induced by major defects in lipid metabolism.

Coexpression of herpesviral thymidine kinase reporter gene and VEGF gene for noninvasive monitoring of therapeutic gene transfer: an in vitro evaluation.

UNLABELLED: Coexpression of a reporter gene and a therapeutic gene may allow for noninvasive monitoring of cardiac gene therapy. We sought to evaluate the usefulness of an adenoviral vector expressing mutant herpesviral thymidine kinase reporter gene (HSV1-sr39tk) and vascular endothelial growth factor (VEGF) 121 in independent expression cassettes (Ad4tk). METHODS: Accumulation of 14C-2'-fluoro-5-methyl-1-beta-D-arabinofuranosyluracil (FIAU) and 9-(4-18F-fluoro-3-hydroxymethylbutyl)guanine (FHBG) as reporter probes, and secretion of VEGF into medium, were determined for Ad4tk-infected H9c2 rat cardiac cells in vitro. RESULTS: In vitro tracer uptake increased with increasing vector concentration and over time. It was comparable to cells infected with adenovirus expressing only wild-type HSV1-tk (reporter probe: 14C-FIAU) or mutant HSV1-sr39tk (reporter probe: 18F-FHBG). No significant uptake was observed in cells infected with adenovirus expressing VEGF alone. With increasing vector concentration, Ad4tk-infected cells increasingly released VEGF into medium. VEGF production correlated significantly with cellular reporter probe uptake (r = 0.93; P = 0.0003). CONCLUSION: The usefulness of a vector coexpressing HSV1-tk and VEGF for noninvasive imaging of expression of a therapeutic transgene has been demonstrated in vitro. This approach may allow for future in vivo monitoring of cardiac angiogenesis gene therapy.

Cytosolic delivery of viral nucleoprotein by listeriolysin O-liposome induces enhanced specific cytotoxic T lymphocyte response and protective immunity.

Cytotoxic T lymphocytes (CTLs) are capable of conferring protection against intracellular pathogens and tumor. Protective antiviral immunity, mediated by the activation of antigenic epitope-specific CTL, can be achieved by delivering exogenous antigen into the cytosol of antigen-presenting cells. Cytosolic introduction of vaccine antigen, however, requires a specialized delivery strategy due to the membrane barrier limiting the access of macromolecules to the cytosol. In this study, we have investigated the potential ability of listeriolysin O-containing liposomes (LLO-liposomes) to deliver lymphocytic choriomeningitis virus (LCMV) nucleoprotein (NP), harnessing the intracellular invasion mechanism of Listeria monocytogenes, to stimulate a NP-specific CTL response. We have analyzed the ability of LLO-liposomes to induce an enhanced CTL response and determined the extent of CTL-mediated protection using an in vivo infection model. Mice immunized with LLO-liposomes containing NP generated a higher frequency of NP-specific CD8+ T cells with greater effector activity than the control groups immunized with either non-LLO-liposomal NP or LLO-liposomes containing control protein. Moreover, LLO-liposomal NP-immunized mice were completely protected against a lethal intracerebral challenge with a virulent strain of LCMV and were capable of clearing a chronic LCMV infection. Our study demonstrates that LLO-liposomes can be used as an efficient vaccine delivery system carrying a viral antigenic protein to generate protective antiviral immunity.